The following process has been known as one example of the use of ETI for the purification of tPA. Namely, the purification process comprises providing human melanoma cells, causing the cells to produce tPA in a serum-free medium, harvesting the culture crop, charging it into an ETI column to adsorb tPA, and then eluting ETI-adsorbed tPA with a 1-3M aqueous solution of potassium thiocyanate (Japanese Patent Laid-Open No. 118717/1984). This purification process is however a process for purifying tPA but does not relate to the separation and purification of single-chain tPA and double-chain tPA.
The present inventors have already developed inter alia a process for separating and removing tPA which occurs in a culture medium containing fetal calf serum, reacts to anti-human tPA antibody and has a molecular weight of 110,000.+-.20,000 daltons as well as a process for culturing cells, in which tPA gene has been integrated by using recombinant DNA technology, and then separating tPA derived from host cells and tPA originated from human cells (Japanese Patent Laid-Open No. 168601/1985).
Substances having the above tPA activity include both single-chain and double-chain ones. It has been found that both of them have a molecular weight of about 70,000 daltons but the single-chain tPA and double-chain tPA are different in plasminogen activating ability and affinity to fibrins. Namely, it has been uncovered that double-chain tPA has plasminogen activating ability as high as about ten times compared with single-chain tPA (Japanese Patent Laid-Open No. 118717/1984) and on the other hand, single-chain tPA has greater affinity to fibrins compared with double-chain tPA and upon adsorption on fibrins, is converted very quickly into double-chain tPA. Single-chain tPA is therefore preferable for obtaining the desired activity to the maximum extent at the site of clots [D. C. Rijken, et al., J. Biol. Chem., 257, 2920-2925 (1982)].
As a method known for obtaining single-chain tPA, it has been known to add a proteolytic enzyme inhibitor upon culture of cells so as to inhibit the conversion from single-chain tPA into double-chain tPA (D. C. Rijken, et al., J. Biol. Chem., 256, 7035-7041 (1981)]. This method is however very difficult to suppress the conversion into double-chain tPA completely under widely-varying conditions such as the kind of cells, the manner of cell cultivation and the cycle of cultivation. Namely, the kind and concentration of a proteolytic enzyme contained in a culture medium for cells generally differ from one medium to another and considerable difficulties are involved in controlling the action of such proteolytic enzymes. On the other hand, there is a wide variety of proteolytic enzyme inhibitors, including those giving adverse influence to the multiplication of cells. A limitation is therefore imposed on the kind of proteolytic enzyme inhibitors which are usable in the cultivation of cells. Furthermore, some kinds of proteolytic enzyme inhibitors are expensive and are hence impractical for use in cultivation on a large scale.
As a method for obtaining single-chain tPA in a pure form, it has also been known to use an immobilized monoclonal antibody which adsorbs single-chain tPA specifically (catalogue of Biopool AB, Sweden). This method is however accompanied by problems in the adsorbability to the immobilized monoclonal antibody and the stability of a column of the antibody during its use.